REGULATION OF LACTATE DEHYDROGENASE AND

GLYCEROL-3-PHOSPHATE DEHYDROGENASE IN

MAMMALIAN HIBERNATION

Anthony A. Ruberto

B.A., 2013

Hamilton College (NY)

A Thesis Submitted to the Faculty of Graduate Studies and Research in partial fulfillment

of the requirements for the degree of

Master of Science

Department of Biology

Carleton University

Ottawa, Ontario, Canada

© Copyright 2015

Anthony A. Ruberto

II

The undersigned hereby recommend to the Faculty of Graduate Studies and Research

acceptance of this thesis

“REGULATION OF LACTATE DEHYDROGENASE AND GLYCEROL-3-

PHOSPHATE DEHYDROGENASE IN MAMMALIAN HIBERNATION”

submitted by

Anthony Agostino Ruberto, B.A.

in partial fulfillment of the requirements for the degree of Master of Science

____________________________________

Chair, Department of Biology

___________________________________

Thesis Supervisor

Carleton University

III

Abstract

Hibernation is a winter survival strategy for many small mammals. Animals sink into deep

torpor, body temperature falls to near 0°C and physiological functions are strongly

suppressed. Enzymes are the catalysts of metabolic pathways in cells and their appropriate

control is critical to hibernation success. This thesis explores the properties and regulation

of two key enzymes of carbohydrate metabolism (lactate dehydrogenase, LDH) and lipid

metabolism (glycerol-3-phosphate dehydrogenase, G3PDH) purified from liver and

skeletal muscle of ground squirrels (Urocitellus richardsonii). The studies showed that

changes in pH, temperature, and inhibitors play roles in differentially regulating these

enzymes between euthermic and torpid states. Furthermore, reversible protein

phosphorylation proved to be a significant regulatory mechanism, producing a reduced

activity state of skeletal muscle LDH and increased activity state of G3PDH in both skeletal

muscle and liver during torpor. Overall, these studies showed that multiple mechanisms of

enzyme regulation, particularly protein phosphorylation, contribute to reorganizing fuel

metabolism during hibernation.

IV

Acknowledgements

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Sleep fast, carpe diem, no regrets. Jones out.

V

Table of Contents Page

Title Page I

Acceptance Sheet II

Abstract III

Acknowledgements IV

Table of Contents V

List of Abbreviations VI

List of Figures IX

List of Tables XI

List of Appendices XII

Chapter 1 General Introduction 1

Chapter 2 Purification and Properties of Lactate Dehydrogenase 17

from the Skeletal Muscle of the Hibernating Ground

Squirrel, Urocitellus richardsonii

Chapter 3 Purification and Properties of Glycerol-3-Phosphate 48

Dehydrogenase from the Liver of the Hibernating

Ground Squirrel, Urocitellus richardsonii

Chapter 4 Purification and Properties of Glycerol-3-Phosphate 81

Dehydrogenase from Skeletal Muscle of the Hibernating

Ground Squirrel, Urocitellus richardsonii

Chapter 5 General Discussion 105

References 112

Appendices 125

VI

List of Abbreviations

β-GP β-glycerophosphate

β-MeSH β-mercaptoethanol

ADP Adenosine diphosphate

ANOVA One-way analysis of variance

AMP Adenosine monophosphate

AMPK AMP-activated protein kinase

AP Alkaline phosphatase

ATP Adenosine triphosphate

BAT Brown adipose tissue

CaMK Calcium-calmodulin dependent kinase

cAMP 3'-5'-cyclic adenosine monophosphate

cGMP 3'-5'-cyclic guanosine monophosphate

CM- Carboxymethyl

DEAE+ Diethylaminoethyl

DHAP Dihydroxyacetone phosphate

Ea Activation energy

ECL Enhanced chemiluminescence

EDTA Ethylenediamine-tetraacetic acid

EGTA Ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid

FA Fatty acid

FABP FA binding protein

FAS Fatty acid synthase

G3P Glycerol-3-phosphate

G3PDH Glycerol-3-phosphate dehydrogenase

GAPDH Glyceraldehyde-3-phosphate dehydrogenase

GK Glycerol kinase

GuHCl Guanidine hydrochloride

HA Hydroxyapatite

VII

HK Hexokinase

HSL Hormone-sensitive lipase

IC50 Concentration producing half-maximal inhibition

Km Substrate concentration producing half-maximal enzyme velocity

LDH Lactate dehydrogenase

MOE Molecular Operating Environment

MP Melting point

MR Metabolic rate

NADH β-Nicotinamide adenine dinucleotide, reduced form

NST Non-shivering thermogenesis

PAGE Polyacrylamide gel electrophoresis

PDH Pyruvate dehydrogenase

PDK4 PDH kinase isoenzyme 4

PI3K Phosphoinositide-3 kinase

PK Pyruvate kinase

PKA Cyclic AMP dependent protein kinase A

PKG Cyclic GMP dependent protein kinase G

PMSF Phenylmethylsulfonyl fluoride

PP1 Protein phosphatase 1

PP2A Protein phosphatase 2A

PP2B Protein phosphatase 2B

PP2C Protein phosphatase 2C

PTL Pancreatic triacylglycerol lipase

PTM Post-translational modification

PUFA Polyunsaturated fatty acid

PVDF Polyvinylidene difluoride membrane

Q10 Ratio of the rate of a reaction at one temperature divided by the rate at a temperature 10°C less

RGS Richardson’s ground squirrel

RPP Reversible protein phosphorylation

VIII

RQ Respiratory quotient

SDS Sodium dodecyl sulfate

T50 Temperature at which half of the protein is irreversibly denatured

Tb Body temperature

TBS Tris-buffered saline

TBST Tris-buffered saline containing the detergent Triton-X

TKin Total endogenous kinases

TPPase Total endogenous phosphatases

WAT White adipose tissue

13-LGS 13-Lined ground squirrel

IX

List of Figures

Page

Figure 1.1. Body temperature as a function of time over one year 16

starting in June for a golden-mantled ground squirrel

(Callospermophilus lateralis).

Figure 2.1. Typical Cibacron Blue elution profiles for LDH activity 37

from muscle of euthermic (control) and hibernating

U. richardsonii.

Figure 2.2. 10% SDS-PAGE gel with silver staining of samples taken 38

at each step in the purification of LDH from muscle of

euthermic U. richardsonii.

Figure 2.3. Effects of in vitro incubations to stimulate the activities of 39

endogenous (a) protein kinases or (b) protein phosphatases

on the Km for L-Lactate for LDH from euthermic

U. richardsonii muscle.

Figure 2.4. Quantification of post-translational modifications of 40

Purified LDH from muscle of euthermic and hibernating

U. richardsonii.

Figure 2.5. Michaelis-Menten curves for (a) forward (lactate-utilizing, 41

5˚C) and (b) reverse (pyruvate-utilizing, 5˚C) reactions

catalyzed by purified euthermic and hibernating LDH; and (c)

Arrhenius plots for the forward reaction LDH at three

temperatures: 5˚C, 22˚C, 37˚C.

Figure 3.1. Typical elution profiles for G3PDH activity from liver of 65

euthermic (control) and hibernating U. richardsonii.

Figure 3.2. 10% SDS-PAGE gel with silver staining of samples taken at 66

each step in the purification of G3PDH from liver of euthermic

U. richardsonii.

Figure 3.3. Quantification of post-translational modifications of purified 67

G3PDH from liver of euthermic and hibernating U. richardsonii

via (a) phosphorylation, and (b) methylation, acetylation, and

ubiquitination.

X

Figure 3.4. The predicted structure of G3PDH from liver of hibernating 68

Richardson’s ground squirrel using SWISS-MODEL with human

G3PDH as reference (PDB identifier: 1x0v.pdb1.gz).

Figure 3.5. Michaelis-Menten curves for substrates of the G3P-utilizing 69

(22˚C, pH 8.0) reaction catalyzed by purified euthermic (a,b) and

hibernating (c,d) G3PDH.

Figure S3.1. Amino acid alignment of thirteen-lined ground squirrel G3PDH 70 cytoplasm isoform X1 (tVariant1; Accession: XP_005324992.1)

with isoform X2 (tVariant2; Accession: XP_005324993.1). Figure S3.2. Multiple alignment of the deduced amino acid sequence of 71

thirteen-lined ground squirrel (13LGS; I. tridecemlineatus) G3PDH as compared to G3PDH sequences from three non-hibernating mammalian species.

Figure S3.3. Predicted phosphorylation sites for 13-LGS G3PDH cytoplasm 72

(a) isoform X1 and (b) isoform X2 using NetPhos 2.0 server (Blom et al., 1999).

Figure S3.4. Crystal structure of human glycerol-3-phosphate dehydrogenase 73

1-like protein (PBD ID: 1X0V).

Figure S3.4. Kinetic values obtained from multiple independent determinations 74 for the G3P-utilizing (22˚C, pH 8.0) reaction catalyzed by purified euthermic (a,b) and hibernating (c,d) G3PDH.

Figure 4.1. Typical elution profiles for G3PDH activity from muscle of 95

euthermic (control) and hibernating U. richardsonii. Figure 4.2. 10% SDS-PAGE gel with silver staining showing FroggaBio 96

molecular weight ladder (kDa) (left) and purified euthermic U. richardsonii skeletal muscle G3PDH (right).

Figure 4.3. Quantification of post-translational modifications of purified 97 G3PDH from muscle of euthermic and hibernating U. richardsonii via phosphorylation.

Figure 4.4. The predicted structure of muscle G3PDH from hibernating 98

Richardson’s ground squirrels using I-TASSER (Yang et al., 2015; Roy et al., 2010; Zhang, 2008) with the 13-LGS G3PDH amino acid sequence as reference (NCBI Reference Sequence: XP_005324992.1).

Figure S4.1. Kinetic values obtained using direct-linear plots (Eisenthal, 1974). 99

XI

List of Tables

Page

Table 2.1. Purification and yield of U. richardsonii LDH from muscle 34 of (a) euthermic and (b) hibernating ground squirrels. Table 2.2. Kinetic parameters of purified muscle LDH from euthermic 35

and hibernating U. richardsonii.

Table 2.3. Effects of pH on purified muscle LDH from (a) euthermic 36 and (b) hibernating U. richardsonii.

Table 3.1. Purification and yield of U. richardsonii G3PDH from liver 62 of (a) euthermic and (b) hibernating animals.

Table 3.2. Kinetic parameters of purified liver G3PDH from euthermic 63

and hibernating U. richardsonii.

Table 3.3. Effects of pH on purified liver G3PDH from (a) euthermic 64

and (b) hibernating U. richardsonii.

Table 4.1. Purification and yield of U. richardsonii G3PDH from skeletal 92

muscle of (a) euthermic and (b) hibernating.

Table 4.2. Kinetic parameters of purified muscle G3PDH from euthermic 93

and hibernating U. richardsonii.

Table 4.3. Effects of pH on purified muscle G3PDH from (a) euthermic 94

and (b) hibernating U. richardsonii.

XII

List of Appendices

Page

Appendix I Communications at Scientific Meetings 125

Appendix II R you ready for this? A Biochemist’s Guide to Enzyme 127

Kinetic Parameter Analyses Using the Programme R

Chapter 1

General Introduction

2

Mammalian Hibernation

The ability of mammals to produce internal heat for the maintenance of an ideal

and constant body temperature (Tb) has allowed them to survive in some of the most

extreme environments. Surely, the independence that these endotherms have from

restrictions imposed by the thermal environment is quite advantageous. However, the

maintenance of a metabolically favourable temperature comes with a high energetic cost

and, during times of low food availability, such energy demands may not be met,

particularly when cold environmental temperatures create high demands for

thermogenesis. A coping strategy that various species of mammals have developed over

evolutionary time is hibernation, a hypometabolic state where physiological and

biochemical activities are suppressed and energy expenditure is minimized.

Hypometabolism

Although a wide range of invertebrates and vertebrates are capable of entering a

hypometabolic state of suspended animation when environmental conditions are too harsh

for normal function and behaviour (Storey & Storey, 2004, 2007), perhaps the most

complex form of natural hypometabolism is mammalian hibernation. This remarkable feat

is marked not only by entrance into a torpid state but also by the inhibition of

thermogenesis, allowing Tb to fall to near ambient temperature. Whether it be seasonal

torpor (hibernation) where a species undergoes multiple bouts of multi-day torpor over the

winter, or daily torpor—induced at any time of the year by adverse conditions, there is a

degree of phenotypic and metabolic plasticity that hibernating species endure. For clarity

3

purposes, the information that follows is in reference to the seasonal form of torpor,

hibernation.

Some common mammals that exhibit hibernation include genera such as marmots

and woodchucks (Marmota), hedgehogs (Erinaceous), bats (Eptesicus and Myotis), and

ground squirrels (Ictidomys and Urocitellus). As mentioned previously, seasonal torpor is

comprised of multiple torpor bouts lasting several days or weeks while being interrupted

by short, periodic arousals (French, 1988) (Fig.1.1). Current theories as to why hibernators

awake from dormancy for short periods of time throughout the hibernating season are

described below in section iv. Interbout arousal.

Phenotypic plasticity associated with hibernation

i. Key Physiological Changes

Entry in to torpor is marked a decrease in heart rate, a decrease in respiratory rate,

and an increase in vasoconstriction to maintain proper blood pressure, all while the

hypothalamic temperature set point is reset to allow Tb fall at a progressive, controlled rate

(Carey et al., 2003). Once in torpor, Tb is maintained near ambient temperature (Carey et

al., 2003), while physiological functions such as heart rate and breathing rate may be

reduced to as little as 3% (Zatzman, 1984) and 1% (McArthur and Milsom, 1991),

respectively, relative to euthermic values. Along with these physiological adaptations

during torpor, there is a reduction in immune function (Prendergast et al., 2002) and a

reduction in metabolic rate to as low as just 1-5% of the corresponding euthermic rate

(Geiser, 2004). Torpor arousal—whether it be interbout or exit out of hibernation at the

end of the season—is dynamic (much quicker than torpor entry) and is usually

4

accomplished within 20-30 minutes in small rodents. During this time, Tb rises, initially

driven via non-shivering thermogenesis (NST) in brown adipose tissue (BAT), and then

skeletal muscle shivering is also initiated after Tb reaches ~20°C; high rates of

thermogenesis continue until the animal reaches normothermy, substrates are mobilized

for energy production, and the cardiovascular system is stimulated (Carey et al., 2003).

ii. Pre-hibernation adjustments

Despite the fact that basal metabolic rates are reduced significantly during

hibernation, species must build up sufficient fuel reserves to last them through the

hibernating season which is often more than half the year. Some species opt to store food

in their burrows and eat during interbout arousals, however many do not. For the latter

individuals, hibernation is effectively a state of long term starvation and therefore

metabolic adjustments must be made to meet essential energy demands. Hibernators, such

as marmots and ground squirrels, go through a period of intense eating (hyperphagia) in

the late summer and early fall months to prepare themselves for the hibernation season

(Davis, 1976). During this period, body mass is increased by approximately 50% largely

due to the deposition of triglycerides in white adipose tissue (WAT). Elevated activities of

lipogenic enzymes support this preparatory phase. For example, fatty acid synthase (FAS)

activity in hibernator lipogenic tissue is highest in the prehibernating phase, suggesting

high rates of fatty acid (FA) synthesis (Anderson et al., 1989; Turner et al., 1989; Mostafa

et al., 1993).

During the prehibernating fattening period, hormonal controls on lipid storage and

satiety are modified. An important hormone that is associated with the buildup of adipose

5

tissue is leptin, the satiety hormone. This hormone serves as a feedback controller by

signaling the hypothalamus with information about body fat levels and energy balance

(Ahima et al., 1996). Produced and secreted by adipose cells, its levels are normally

positively correlated with body adiposity (Considine et al., 1996; Maffei et al., 1995).

Because adiposity is expected to increase before hibernation, an increase in leptin would

serve as a problem for hibernators since this hormone generally decreases food intake,

increases energy expenditure, and decreases body mass. However, hibernating species are

capable of building adipose tissue while circumventing the effects of leptin. Opposite of

the normal response to increasing adiposity in nonhibernating species, research by

Kronfeld-Schor et al. (2000) revealed that leptin secretion and sensitivity decreased during

the prehibernating phase in little brown bats (Myotis lucifugus). These findings suggest that

during the prehibernatory phase these animals somehow overcome the satiety and

metabolic signals associated with leptin to deposit large fat stores.

Another key hormone that aids in the prehibernating fat storage period is insulin.

As hyperphagic mammals ingest food, the pancreas secretes insulin—the key metabolic

hormone that stimulates circulating blood glucose uptake, promotes glycogen anabolism,

and stimulates the storage of excess fuel as fat. Previous research has shown that prior to

hibernation, insulin levels are elevated and then fall during hibernation (

6

lipase (HSL) activity and thereby facilitate the accumulation of triglycerides (Stralsfor and

Honner, 1989).

Along with modifications that promote fat storage, hibernators must be able to

mobilize and utilize lipids at low Tb. Since lipid depots in most mammals have melting

points (MPs) around 25°C, these lipids would be solid and therefore be difficult to utilize

at hibernating Tb (Wang, 1979). With this in mind, lipid fluidity—which is largely

dependent on the lipid degree of unsaturation (unsaturation is proportional to fluidity)—is

an important factor in determining hibernation success. By eating a diet rich in mono

unsaturated (ie. oleic acid [18:1]) and polyunsaturated fatty acids (PUFAs) (ie. linoleic acid

[18:2] and α-linolenic acid [18:3]) which have MPs in the range of 5 and -6.5°C,

hibernating species are capable of incorporating these lipids into their triglycerides to

create adipose fuel depots that can be mobilized over the wide range of Tb values that they

endure during their torpor-arousal periods (Harlow and Frank, 2001, Frank et al., 2008).

Lipids are particularly important as fuels for the intense periods of thermogenesis by BAT

and skeletal muscle that are necessary to rewarm the hibernator body to euthermia. Many

questions still remain unexplored regarding lipid fluidity. For instance, the natural sensing

mechanism for fat depot PUFA content remains unknown as well as how the fluidity of

phospholipids in membranes is adjusted to maintain functionality at the low Tb values

endured during hibernation.

During the brief interbout arousal phases of hibernators, carbohydrate oxidation

reasserts itself as a main source of fuel for energy production in most tissues as indicated

by a respiratory quotient (RQ; the ratio of the volume of carbon dioxide eliminated to the

volume of oxygen consumed by an animal) that increases to a value near 1. As reported by

7

Carey et al. (2003), RQ values during hibernation fall to a value closer to 0.7, indicating a

switch to lipid oxidation to meet energy demands. Given the fact that during arousal RQ

values are around 1 and, that protein catabolism is unfavourable for energy production, it

is expected that proteolysis and amino acid catabolism is generally limited throughout

hibernation.

iii. Hibernation

Successful hibernation is sensitive not only to the quantity of stored fat but also the

quality of stored fat. Various studies have shown that torpor-arousal cycles either do not

occur or are disturbed if fat stores are insufficient or if the amounts of necessary fatty acids

are too low (Cranford, 1978; Geiser et al., 1994; Frank, 1992; Florant, 1992; Florant et al.,

1993). Other than tissues that are highly specialized to use carbohydrates as fuels (e.g.

brain, red blood cells), most carbohydrate metabolism is spared in hibernator organs during

torpor. The switch from carbohydrate to lipid metabolism has been confirmed via studies

measuring glycolytic, lipogenic, and lipolytic enzyme activities. First, research by Brooks

and Storey (1992) suggested that the covalent modification of liver glycogen

phosphorylase in the golden-mantled ground squirrels (Callospermophilus lateralis)

during hibernation decreased enzymatic activity and thereby limited the amount of

available glucose in blood. Furthermore, this research found that pyruvate dehydrogenase

(PDH) activity in heart and kidney was dramatically decreased during hibernation,

implicating a strong inhibition of carbohydrate oxidation during torpor. Secondly, research

by Frank et al. (1998) showed that liver FAS activity in the black-tailed prairie dog

(Cynomys ludovicianus) is reduced during hibernation, limiting the overall rate of fatty

acid synthesis. Last, the restoration of HSL activity, along with the activity of pancreatic

8

triacylglycerol lipase (PTL) in WAT during hibernation further supports the importance of

lipids for energy production while torpid (Bauer et al., 2001). Together, these findings

suggest that glycolysis is suppressed and there is a greater dependence on fuel metabolism

based primarily on lipid oxidation during hibernation.

During hibernation, blood glucose and insulin levels are reduced and are lowest

during mid-winter due to halted food intake during dormancy (Bauman et al., 1987).

Previous research has shown that the phosphoinositide-3-kinase (PI3K)-Akt pathway is

mediated by insulin binding to insulin receptors and during hibernation, as insulin levels

decrease, PI3K mediated activation of Akt is suppressed (Cai et al, 2004, Abnous et al.,

2008). The resulting downstream events of suppressed Akt signaling include decreases in

both glycogen synthesis and lipogenesis (Porstmann et al. 2008; Laplante and Sabatini,

2010). The drop in insulin levels and subsequent decrease in glucose uptake by tissues,

combined with the down regulation of several glycolytic enzymes, serve as important

factors in the reduced glycolytic output during hibernation.

One might expect that to make up for the decrease in plasma glucose levels during

hibernation that glucagon activity would be stimulated. However, previous research in

ground squirrels suggests that glucagon levels remain constant in hibernators (Bauman et

al., 1987). Furthermore, this finding suggests that the plasma glucagon to insulin ratio is

increased during hibernation which may in turn have an influence in the shift of fuel usage

during this period. Most notably, glucagon activates protein kinase A (PKA) that in turn

phosphorylates many metabolic enzymes. For example, glucagon acts on WAT to active

PKA mediated phosphorylation of HSL (Moreau-Hamsany et al., 1988). The activation of

PKA by glucagon also leads to the phosphorylation and subsequent inhibition of pyruvate

9

kinase (PK) (Pilkis and Claus, 1991). Furthermore, glucagon inhibits PK mRNA

transcription as well as increases degradation of existing PK mRNA (Pilkis and Claus,

1991). The combination of glycolytic suppression and lipolysis activation during

hibernation leads to an increase in circulating plasma FAs and facilitates the reorganization

of fuel metabolism to favour a heavy reliance on lipid catabolism.

iv. Interbout Arousal

Much of the stored fuel used by hibernators is consumed for thermogenesis during

the periodic arousals. It is estimated that the re-warming process and the time spent in short

periods of arousal consume approximately 40-70% of the energy budget for the whole

hibernation season (Wang, 1979). As stated previously, lipid oxidation is thought to

mediate the initial steps of the rewarming process, with increased reliance on carbohydrates

usage as Tb rises. A shift in RQ from 0.7 to >0.85 during the short interbout arousal periods

supports the idea of increased carbohydrate usage (Buck and Barnes, 2000; Karpovich et

al., 2009). Furthermore, interbout arousal periods occur in all hibernating species and are

essential for survival; however, their function is still debated. Current theories as to why

periodic arousals occur include: the necessity to restore metabolic imbalances accruing at

low Tb, to restore sleep patterns, and to reactivate the immune system to fight any

pathogens that have accumulated during hibernation (French, 1985; Daan et al., 1991;

Prendergast et al., 2002).

Reversible Post-translational Modifications

Many cellular proteins are altered by covalent modifications after they are

translated. The covalent addition of different functional groups to a protein alters its

structural properties and may influence its functionality also. With more than 30 post-

10

translational modifications (PTMs) known to date, the combinatory effects of various

PTMs make the proteome one of the most dynamic biological systems.

Probably the most studied post-translational modification in mammals is reversible

protein phosphorylation (RPP). RPP is a major mechanism used for regulatory control

proteins involved in virtually every cellular process (Storey and Storey, 2004a). Protein

kinases and protein phosphatases mediate the addition and removal of a phosphate group,

respectively, to specific serine, threonine or tyrosine residues of a target protein. In this

way, fine tuning by phosphorylation/dephosphorylation serves as quick, energetically

cheap (relative to the cost of gene expression) method that can have profound effects on a

protein’s biological activity, interaction with other proteins, stability, movement between

subcellular compartments, or cellular fate (Cohen, 2002). The dynamic nature of this

process helps explain why almost all eukaryotic cells utilize reversible phosphorylation as

a general regulatory mechanism. Studies in our lab have demonstrated that reversible

phosphorylation is a crucial regulatory mechanism in animal responses to several types of

environmental stress (Storey and Storey, 2004a), including mammalian hibernation

(MacDonald and Storey, 1999; Bell and Storey, 2010; Bell et al., 2014). For example, RPP

alters the activity of Na+/K+ATPase to help reduce energy expenditure during global

metabolic rate depression (MacDonald and Storey, 1999), controls PDH activity to regulate

carbohydrate oxidation (Tessier et al., 2015), regulates amino acid metabolism at glutamate

dehydrogenase (Thatcher and Storey, 2001; Bell and Storey, 2010), regulates multiple cell

signaling cascades and transcription factors (Luu et al., 2014; Cowen and Storey, 2003;

and reviewed in Tessier and Storey, 2014), contributes to cell cycle suppression (Wu and

Storey, 2012), etc.

11

Model Animal

Richardson’s ground squirrel (Urocitellus richardsonii) is the hibernator model

used in this thesis. Also known as prairie gophers, this species can be found in portions of

Manitoba, Saskatchewan, Alberta, North Dakota, South Dakota, Montana, and Minnesota.

The diet of Richardson’s ground squirrels consists of seeds, insects, flowers, and leaves.

The weight of these ground-dwelling, burrowing mammals varies between 0.2 and 0.4

kilograms when they are euthermic and as high as 0.75 kilograms when they enter

hibernation. Their meandering burrows are approximately 3.5 inches in diameter, 4-5 feet

below the surface, and span 15-20 meters in length. Richardson’s ground squirrels

hibernate alone in a chamber called a hibernaculum. The hibernation season of these

species varies between males and females since the males generally come out of torpor 8-

16 days earlier than female squirrels (Michener, 1983); however, on average, this species

hibernates for 7-9 months, beginning in September and ending as late as May. My research

in this thesis is focused on enzyme regulation in liver and skeletal muscle tissue of

Richardson’s ground squirrels comparing euthermic and torpid animals to assess changes

the enzyme properties as a function of hibernation.

Target tissues

Skeletal Muscle

Both long periods of inactivity and limited fuel availability are two problems that

skeletal muscle must manage when a species enters a hibernating state. In non-hibernating

species (including man), long periods of inactivity such as occur during torpor would lead

to significant atrophy of skeletal muscle. However, hibernators experience much less

12

muscle atrophy during torpor than would be expected from such prolonged periods of

inactivity. Research by Wicker et al. (1991), measured muscle mass and levels of an

enzyme whose activity reflects aerobic capacity, citrate synthase (CS), in hibernating

golden-mantled ground squirrels. Their results indicated that mass-specific activity of this

enzyme increased in the hibernating state (Wicker et al., 1991). Although CS may not

contribute to strength of skeletal muscle directly, it serves to help preserve mechanisms

associated with heat production required during the arousal rewarming process (Wicker et

al., 1991). As shivering thermogenesis commences during the rewarming process in

hibernators, heart rate drastically increases. With that in mind, muscles that are well

supplied with blood oxygen and both white and red fibers contribute to heat production

(Ambid and Agid, 1975; Feist, 1970; Lyman, 2013). Research by Postnikova et al. (1999)

revealed that a very high myoglobin content in muscle of hibernators relative to their non-

hibernator counterparts can contribute to the explosive rewarming process during arousal.

Furthermore, to support muscle contractility and shivering thermogenesis during

hibernation, both of which involve ATP hydrolysis by myosin ATPase, Fahlman et al.

(2000) reported hibernation-responsive up-regulation of the transcript levels of myosin

light chain 1 (MLC1v) in skeletal muscle of hibernating golden-mantled ground squirrels.

Up-regulation of the expression of gene products coding for metabolic proteins also occurs

in skeletal muscle during hibernation. The up-regulation of FA binding protein (FABP)

and PDH kinase isoenzyme 4 (PDK4) suggest that hibernator skeletal muscle relies on

enhanced lipid metabolism during torpor (Hittel and Storey, 2001; Buck et al., 2002).

These data indicate that adaptive changes to selected cellular functions in skeletal muscle

are required for successful hibernation.

13

ii. Liver

The liver is a vital organ that plays a major role in coordinating and regulating a

wide range of functions that affect the whole body—these include detoxification reactions,

protein synthesis, glycogen storage, and hormone production. It should be expected that

during hibernation, important metabolic changes occur in liver tissue to meet new cellular

demands. Many of the lipolytic enzymes and proteins involved FA transport are

synthesized in the liver. Regulation of these proteins is essential as hibernators rely largely

on the triacylglycerol fuels stored in adipose tissue during winter dormancy. Like in

hibernator skeletal muscle, FABP activity and expression increases during torpor in

hibernator liver (Stewart et al., 1998; Epperson et al., 2004).

Hypotheses

Given the well documented changes in carbohydrate and lipid metabolism between

various stages of hibernation, I propose that the metabolism of these fuels is regulated at

the enzyme level in Richardson’s ground squirrel skeletal muscle and liver. Furthermore,

I propose that reversible post-translational modification which serves as a rapid,

energetically feasible mechanism of enzyme control plays an important role in optimizing

the structural and functional properties of metabolic enzymes as they switch between

euthermic and hibernating states. Two main hypotheses guide the research in this thesis:

1. In a depressed metabolic state, Richardson’s ground squirrels will modify the

key glycolytic enzyme, LDH, in a way that decreases its enzymatic activity.

14

2. In a depressed metabolic state, glycerol-3-phosphate dehydrogenase (G3PDH),

a key enzyme connecting lipid metabolism and carbohydrate metabolism, will

be activated as indicated by an increase in enzymatic activity.

Objectives

The torpor bouts that occur during the hibernation season are highly regulated

events that not only include physiological changes but also metabolic plasticity to support

long term survival at low Tb in the absence of food intake. One aspect of metabolic

regulation in a mammalian hibernator that has not been studied is the role that post-

translational modifications can play in the differential function of central dehydrogenase

enzymes between euthermic and hibernating states. Research in this thesis explores this by

investigating the structural, functional, and regulatory properties of skeletal muscle LDH

and liver and muscle G3PDH. LDH converts pyruvate and (reduced β-Nicotinamide

adenine dinucleotide) NADH, two products of glycolysis, to lactate and NAD+ when

oxygen is absent or in short supply. Chapter 2 presents an analysis of this important enzyme

associated with carbohydrate metabolism to provide us with a more clear understanding of

the state of glycolysis during ground squirrel hibernation.

G3PDH is an important enzyme that connects lipid metabolism to carbohydrate

metabolism— by catalyzing the reversible conversion of glycerol-3-phosphate (G3P) and

NAD+ to dihydroxyacetone phosphate (DHAP) and NADH. With increase importance of

lipid catabolism during hibernation, examination of this enzyme is of interest to determine

its potential differential regulation during ground squirrel torpor. Studies of this enzyme

from liver and muscle of euthermic and hibernating ground squirrels are present in

Chapters 3 and 4, respectively.

15

Overall, the studies in this thesis are aimed to elucidate the structural and functional

properties of key metabolic enzymes in skeletal muscle and liver during hibernation. A

greater understanding of how these enzymes are regulated can add to previous research

conducted on the metabolic adaptations that hibernating species undergo as part of this

unique mammalian survival strategy.

16

Figure 1.1. Body temperature as a function of time over one year starting in June for a

golden-mantled ground squirrel (Callospermophilus lateralis). Inset depicts the entrance

into torpor (EN), early torpor (ET), late torpor (LT), arousal (AR), and interbout arousal

(IBA) (Taken from Carey et al., 2003).

Chapter 2

Purification and Properties of Lactate

Dehydrogenase from the Skeletal Muscle

of the Hibernating Ground Squirrel,

Urocitellus richardsonii

18

Introduction

Lactate dehydrogenase (LDH; E.C. 1.1.1.27), the terminal enzyme of anaerobic

glycolysis, catalyzes the following reversible reaction:

L-lactate + NAD+ ↔ pyruvate + NADH + H+

LDH plays a critical role in energy metabolism as it facilitates the production of ATP via

glycolysis during low oxygen conditions by recycling the essential coenzyme NAD+

(nicotinamide adenine dinucleotide). Skeletal muscle (particularly white muscle fibers) is

a tissue that relies heavily on carbohydrate catabolism to produce ATP and power intense

muscle work. Here, the LDH reaction favours lactate formation using the pyruvate and

NADH output of glycolysis (Hochachka and Somero, 2014).

During mammalian hibernation, greater emphasis is placed on lipid oxidation to

meet energy demands, and ATP generation via carbohydrate catabolism is thought to be

suppressed (Buck and Barnes, 2000; Tashima et al., 1970). Mitochondria instead rely on

acetyl-CoA generated from lipid breakdown as the main source of carbon to supply the

Krebs cycle. Previous research has shown that pyruvate dehydrogenase (PDH) is regulated

during hibernation showing heavy suppression to strongly limit entry into the Krebs cycle

by carbohydrate coming from glycolysis (Brooks and Storey, 1992). With this in mind, it

is proposed that other fates for glycolytic carbon could also be suppressed during

hibernation which could include regulation of LDH to help minimize/coordinate the

glycolytic rate and limit lactate buildup in hibernating mammals.

The LDH reaction has long been known to play a major role in cellular redox

balance, being responsible for regenerating the NAD+ needed to allow anaerobic glycolysis

to run. More recently, intracellular NAD+ has been shown to play a major role as a

19

metabolite capable of regulating the transcription of genes associated with metabolism and

circadian rhythms (Cantó et al., 2009; and reviewed in Takahashi et al., 2008; Chaudhary

and Pfluger, 2009). Specifically, new research on NAD-dependent deacetylase sirtuin-1

(SIRT1), a protein that regulates circadian genes (Asher et al., 2008), shows that the

activity of this enzyme is activated by NAD+ (Imai et al., 2000). These discoveries could

tie together cellular nutrient status, metabolic substrate use, and metabolic plasticity during

hibernation.

Richardson’s ground squirrels, Urocitellus richardsonii, have an amazing ability to

survive winter conditions—marked by harsh environmental temperatures and low food

availability—by entering into hibernation. While in this hypometabolic state, core body

temperature (Tb) falls (often to as low as 0–5 °C), and there is a strong suppression of all

physiological processes (e.g. heart rate and breathing rate), suppression of aerobic cellular

respiration, a reprioritization of energy expensive processes (e.g. transcription, translation,

and ion pumping), and reorganization of fuel metabolism (McArthur and Milsom, 1991;

Zatman, 1984; and reviewed in Carey et al., 2003, Staples and Brown, 2008, Storey and

Storey, 2004b; Geiser, 2004).

Reversible phosphorylation of functional proteins and enzymes is a major

regulatory mechanism that mediates the plasticity of metabolic reactions when animals

enter hypometabolic states. This process has been studied extensively in hibernating

species, with ion motive ATPases (MacDonald and Storey, 1999), glycolytic enzymes

(Brooks and Storey, 1992), ribosomal proteins (van Breukelen et al., 2004), and

transcription factors (reviewed in Carey et al., 2003; Storey, 1997) all showing differential

regulation via reversible phosphylation.

20

In this chapter, I reasoned that the transition from active euthermia to a low Tb,

hibernating state should lead to control of LDH in a manner that promotes NAD+

production. Previous research has shown that during entrance in to torpor, NAD+ levels

increase (Serkova et al. 2011). Furthermore, this signaling molecule has been shown to

upregulate proteins that aid in torpor survival (reviewed in Melvin and Andrews, 2009).

Despite an overall decrease in carbohydrate catabolism, maintenance of NAD+ levels could

have profound effects that allow ground squirrels to survive throughout the hibernating

season. Furthermore, such control could contribute to the reorganization of fuel metabolism

to favor lipid oxidation, thereby allowing fat catabolism to contribute to energy needs

during torpor.

In a previous study, Dawson et al. (2013) documented significant differences in

the kinetic and physical properties of purified white muscle LDH from of normoxic versus

anoxic red-eared sliders, Trachemys scripta elegans, due to reversible protein

phosphorylation. The present study demonstrates that ground squirrel skeletal muscle LDH

is also regulated by reversible protein phosphorylation. Furthermore, the changes in the

phosphorylation state of LDH occurring during hibernation make significant changes to

the enzyme’s structural and functional properties.

Materials and Methods

Animals

The protocols used for care and handling of Richardson’s ground squirrels, U.

richardsonii, were as reported previously (MacDonald and Storey, 1998; Thatcher and

Storey, 2001). Briefly, animals were captured in late summer near Calgary, Alberta. All

21

animals were individually housed in rat cages with free access to food and water at 22°C

and on an autumn photoperiod (10 h light, 14 h dark). After 8 weeks under this regime,

half of the animals were maintained under these control (euthermic) conditions. The others

were moved into a 4°C cold room that was maintained in darkness; free access to water

was maintained but food was removed. Squirrels were allowed to enter torpor and animals

were sampled after 2 d continuous torpor (rectal temperature 5-8°C). Euthermic animals

were sampled on the same day. Both hibernating and euthermic animals were killed by

decapitation and tissues were immediately excised, frozen in liquid nitrogen and then

stored in -80°C.

Preparation of tissue extracts

Frozen hind leg skeletal muscle samples were homogenized 1:5 w:v in buffer A (20

mM potassium phosphate, pH 7.2, 1 mM EDTA, 1 mM EGTA, 15 mM β-glycerophosphate

(β-GP), 15 mM β-mercaptoethanol (β-MeSH), 10% v:v glycerol) and a few crystals of

phenylmethylsulphonyl fluoride (PMSF) (added just prior to homogenization) using a

Polytron homogenizer (Brinkmann Instruments, Rexdale, ON, Canada). Homogenates

were centrifuged at 10,000 x g for 30 minutes at 5°C, after which supernatants were

decanted and held on ice until use.

Purification of LDH

A 1.5 mL sample of muscle extract containing ~28 mg of total protein was applied

to a DEAE+ column (1 cm [Diameter] x 5 cm [Height]) previously equilibrated in buffer A. The

column was then washed with 30 mL of buffer A and 4 mL fractions were collected. From

each fraction, a small aliquot was diluted 1:10 v:v in buffer A and then 2 µL from each

22

diluted fraction was assayed to detect LDH activity. The active fractions were pooled and

applied to a Cibacron Blue 3GA (Sigma-Aldrich) column (1 x 5 cm) equilibrated with

buffer A. The Cibacron Blue 3GA column was washed with 30 mL of buffer A to remove

unbound proteins and then a linear gradient of 0–6 mM sodium pyruvate and NADH was

applied to elute bound proteins. Fractions of 1.20 mL were collected and 5 µL from each

fraction was assayed to detect LDH activity. After fractions containing maximal LDH

activity were pooled, low molecular weight metabolites were removed from the sample via

centrifugation (2 min @ 2000 rpm) through small columns of Sephadex G-25 (Sigma-

Aldrich) equilibrated in buffer A. The sample was then loaded on to a second Cibacron

Blue 3GA column (1 x 5 cm) in a similar fashion, although in this case proteins were eluted

with a linear gradient of 0–2 M KCl in buffer A. The fractions were assayed for LDH

activity, and the active fractions were pooled and desalted using an Amicon® Ultra Filter

(Millipore). The purity of the LDH sample was checked by denaturing and reducing gel

electrophoresis followed by silver staining as described below. LDH from the muscle tissue

of euthermic and hibernating ground squirrels were purified in the same manner.

Detection of LDH via Silver Staining Technique

Silver staining procedure was adapted from Blum et al. (1987). In short, after

electrophoresis, the SDS-polyacrylamide gel was incubated for 3 hours in fixing solution

containing 50% (v/v) methanol, 12% (v/v) acetic acid, and 0.05% (v/v) formalin (35%

formaldehyde). Next, the fixative solution was discarded and the gel was washed in 20%

(v/v) methanol (3 x 6 min) before incubating with the sensitizing solution [0.02% (w/v)

sodium thiosulphate (Na2S2O3)] for 2 minutes. The sensitizing solution was discarded and

the gel was washed (2 x 1 min) with ddH20. Next, the gel was incubated in cold silver stain

23

[0.6% (w/v) silver nitrate, 0.076% (v/v) formalin] with gentile shaking for 20 minutes. The

silver stain was then discarded and the gel was washed (2 x 30sec) with ddH20 followed

by addition of developing solution [6% (w/v) sodium carbonate (Na2CO3), 0.04% (w/v)

sodium thiosulphate (Na2S2O3), 0.05% (v/v) formalin] to the gel. Once desired band

intensity on the gel was reached, developing was stopped by adding a terminating solution

[12% (v/v) acetic acid] to the gel. Lastly, bands on the gel were visualized under light and

an image was captured using a ChemiGenius BioImaging system with GeneSnap software

(Syngene, Frederick, MD, USA).

LDH assay

LDH activity was measured as the rate of production or consumption of NADH

using a microplate based assay by measuring absorbance at 340 nm using a Thermo

Labsystems Multiskan spectrophotometer (Thermo Scientific, Waltham, MA, USA).

Optimal assay conditions for LDH in the lactate-oxidizing direction were 30 mM

potassium phosphate buffer (pH 7.5 / 8.0), 60 mM L-lactate, and 2 mM NAD+ in a total

volume of 200 µL with 5 µL of purified enzyme added to start the assay. The optimal

conditions for LDH in the pyruvate-reducing direction were 30 mM potassium phosphate,

1 mM pyruvate, and 0.2 mM NADH in a total volume of 200 µL with 2 µL of purified

LDH. The Km and IC50 values for lactate, NAD+ or pyruvate were determined by holding

the co-substrate constant at 2 mM NAD+, 60 mM lactate, or 0.2 mM NADH. All kinetic

analyses were conducted at 5°C, 22°C, and 37°C in phosphate buffer at pH 7.5 and 8.0.

24

SDS Polyacrylamide Gel Electrophoresis and Immunoblotting

After concentrating 5x using a Amicon® Ultra Filter (Millipore), purified

euthermic and hibernating LDH samples were mixed 1:1 (v:v) with SDS loading buffer

(100 mM Tris buffer, pH 6.8, 4% w:v SDS, 20% v:v glycerol, 0.2% w:v bromophenol blue,

and 10% v:v β-MeSH) and boiled for 5 minutes, cooled on ice and frozen at -20˚C until

use. SDS resolving gels (10% v/v acrylamide, 400 mM Tris, pH 8.8, 0.1% w/v SDS, 0.2%

w/v ammonium persulfate [APS], 0.04% v/v TEMED) were prepared with a 5% stacking

gel (5% acrylamide, 190 mM Tris, pH 6.8, 0.1% w/v SDS, 0.15% w/v APS, 0.1% v/v

TEMED). Purified samples were loaded onto these gels and separated electrophoretically

in SDS-PAGE running buffer (25 mM Tris-base, pH 8.5, 190 mM glycine, and 0.1% w/v

SDS) at 180 V for 45 min. A 2 µL aliquot of protein ladder (FroggaBio) was added to one

lane of every gel to provide molecular weight markers. Commercially purified rabbit

muscle LDH (Boehringer Mannheim) was also loaded onto the gel to confirm the correct

location of LDH subunits. Following electrophoresis, proteins were electroblotted onto

polyvinylidiene difluoride (PVDF) membranes (Millipore) by wet transfer and used for

immunoblotting.

PVDF membranes were equilibrated in methanol before the wet transfer of proteins

from 10% SDS gels. Electroblotting was performed in transfer buffer (25 mM Tris, pH 8.5,

192 mM glycine, and 20% v/v methanol) at room temperature for 1.5 h at 160 mA.

Following protein transfer, PVDF membranes were blocked with 5.0% skim milk for 10

minutes. Membranes were washed three times with Tris-buffered saline (100 mM Tris-

base, 140 mM NaCl, pH 7.6) containing 0.05% Tween-20 (TBST) for 5 min each before

one of the following primary antibodies (all from Invitrogen, Carlsbad, CA, USA) were

25

applied: (1) rabbit anti-phosphoserine (Cat. #618100); (2) rabbit anti-phosphothreonine

(Cat. #718200); (3) rabbit anti-phosphotyrosine (Cat. #615800). All primary antibodies

were diluted 1:1000 v:v in TBST with a small amount of sodium azide added. Primary

antibodies were applied to membranes and allowed to incubate overnight at 4°C with gentle

rocking. Membranes were washed with TBST (3 x 5min), and incubated with a 1:2000 v:v

dilution of goat anti-rabbit IgG-peroxidase secondary antibody for 60 min at room

temperature. Blots were washed with TBST (3 x 5 min) prior to chemiluminescence

visualization on the ChemiGenius Bioimaging System (Syngene, Frederick, MD, USA).

The band intensities were quantified using GeneTools software (Syngene, Frederick, MD,

USA). To confirm equal protein loading of samples, Coomassie blue staining was

subsequently performed on the membranes and used to standardize immunoblot band

intensities. Protein-standardized band intensities for hibernator LDH were then expressed

relative to the standardized signal intensities for control LDH samples.

In vitro Incubations that Stimulate Protein Kinases or Protein Phosphatases

Crude muscle extracts were prepared as described above, and then subsequently

filtered through a small G-50 Sephadex column that had been pre-equilibrated in buffer B

(25 mM potassium phosphate, 10% v:v glycerol, 15 mM 2-mercaptoethanol, pH 7.0).

Aliquots of the filtered supernatants were incubated overnight at 4°C with specific

inhibitors and stimulators of protein kinases or protein phosphatases, as described in

MacDonald and Storey (1999). Aliquot from extracts of both control and hibernator muscle

were mixed 1:2 v:v with the following additions:

26

a. STOP condition (inhibits all protein kinases and protein phosphatases): 2.5 mM

EGTA, 2.5 mM EDTA and 30 mM β-glycerophosphate.

b. Stimulation of total endogenous kinases (TKin): 30 mM β-glycerophosphate, 2

mM AMP, 2 mM cAMP, 10 mM ATP, 2 mM cGMP, 2.6 mM CaCl2, 14 µg/mL

phorbol myristate acetate, 20 mM MgCl2 and 10 mM Na3VO4.

c. Stimulation of total endogenous phosphatases (TPPase): 10 mM MgCl2 and 10

mM CaCl2.

d. Stimulation of endogenous AMP-activated kinase (AMPK): 1 mM AMP, 5 mM

ATP, 10 mM MgCl2, 30 mM β-glycerophosphate, 5 mM Na3VO4.

e. Stimulation of endogenous calcium-calmodulin dependent kinase (CaMK): 1

U of calf intestine calmodulin, 1.3 mM CaCl2, 5 mM ATP, 10 mM MgCl2, 30

mM β-glycerophosphate, 5 mM Na3VO4.

f. Stimulation of endogenous cyclic-AMP dependent protein kinase (PKA): 1

mM cAMP, 5 mM ATP, 10 mM MgCl2, 30 mM β-glycerophosphate, 5 mM

Na3VO4.

g. Stimulation of endogenous cyclic-GMP dependent protein kinase G (PKG): 1

mM cGMP, 5 mM ATP, 10 mM MgCl2, 30 mM β-glycerophosphate, 5 mM

Na3VO4.

h. Stimulation of endogenous protein phosphatases 1 + 2A (PP1+PP2A): 2 mM

EDTA, 2 mM EGTA, 30 mM Na3VO4.

i. Stimulation of endogenous protein phosphatase 2B (PP2B): 2 mM EDTA, 1

µM okadaic acid, 5 mM Na3VO4.

27

j. Stimulation of endogenous protein phosphatase 2C (PP2C): 2 mM EGTA, 1

nM cypermethrin, 1 µM okadaic acid, 5 mM Na3VO4.

After incubation, LDH activities were analyzed in the lactate-oxidizing direction as

described above (30 mM potassium phosphate buffer, pH 8.0, 2 mM NAD+, and varying

lactate) and Km lactate values for LDH were determined and compared to the STOP

condition.

Data Analyses

Maximal LDH activity was determined at 5°C, 22°C and 37°C. The reaction

temperature was altered by placing the Thermo Labsystems Multiskan spectrophotometer

into a VWR International BOD 2020 Incubator (Sheldon Manufacturing Inc., Oregon

USA) set to the desired temperature. Microplates filled with assay mixture (excluding the

enzyme) were equilibrated in the same incubator for several minutes until the desired

temperature was reached (as measured by a telethermometer). Plates were then placed into

the spectrophotometer and reactions were initiated by the addition of enzyme.

Protein concentrations of all samples and fractions were measured using the Bio-

Rad protein assay dye reagent (Bio-Rad, Hercules, CA, USA) with serial dilutions of

bovine serum albumin as the standard according to the manufacturer’s instructions.

Enzyme activities were analyzed with a Microplate Analysis program (Brooks, 1994) and

kinetic parameters were determined using a nonlinear least squares regression computer

program (Kinetics v. 3.51) fully described in (Brooks, 1992). All points were fitted to a

curve determined by the Hill equation (h > 0) within the Kinetics program. Data are

expressed as mean ± S.E.M. from multiple independent determinations of kinetic

parameters on separate preparations of enzyme. Data for euthermic versus hibernating

28

comparisons, pH comparisons, and western blots were analyzed using the Student’s t-test

(two-tailed). Data for kinetic parameters in response to temperature changes were analyzed

using one-way analysis of variances (ANOVAs) followed by a Tukey post hoc test. A

probability value of 95% homogeneity, as determined by gel electrophoresis and staining with silver nitrate

29

(Fig. 2.2). The purified LDH had an apparent subunit molecular weight of 38 kDa (Fig.

2.2).

Reversible phosphorylation of muscle LDH

It was hypothesized that reversible protein phosphorylation might be the

mechanism underlying the kinetic differences between LDH from euthermic and

hibernating muscle reported in Table 2.2. To test this hypothesis, crude muscle extracts

were incubated under conditions that stimulated the actions of endogenous protein kinases

(Fig. 2.3a) or protein phosphatases (Fig. 2.3b) and the resulting effects on an LDH kinetic

parameter (Km lactate) were measured. Incubation of euthermic extracts under conditions

that stimulated the activities of endogenous protein kinases PKG and PKC did not change

LDH Km lactate (Fig. 2.3a). However, stimulation of AMPK, CaMK, or PKA significantly

increased euthermic Km lactate (ie. decreased affinity for lactate). Relative to the STOP

condition (Km lactate = 1.66 ± 0.05mM), AMPK, CAMK or PKA stimulation resulted in

2.0-, 1.7-, and 2.1-fold increases in the Km for crude euthermic LDH, respectively (P

30

residues of hibernating LDH was 2.2 ± 0.30 fold higher compared to euthermic LDH (1 ±

0.07, P

31

lactate-oxidizing and pyruvate-reducing reactions of LDH. Examining the lactate-

oxidizing direction for euthermic LDH, the Ea (34.52 ± 6.68 kJ/mol) was not significantly

different (P>0.05) than the Ea for hibernating LDH (48.61 ± 13.19 kJ/mol, Table 2, Fig.

2.5c). For the pyruvate-reducing direction, comparable value for Ea were 35.10 ± 12.62

kJ/mol for euthermic LDH and 55.26 ± 15.69 kJ/mol, again not significantly different.

In the pyruvate-reducing direction, at 22°C and 37°C there was no difference in the

Km for pyruvate between euthermic and hibernating LDH. However, at 5°C the affinity for

pyruvate was greater (ie. Km lower) for hibernating LDH (0.067 ± 0.005 mM) as compared

to euthermic LDH (0.09 ± 0.01 mM, P

32

Likewise, there was a significant decrease in the Km for NAD+ in the euthermic condition

(Table 2.2). An increase in substrate affinity (decreased Km) was also seen in the

hibernating condition when comparing 37°C to 5°C (Table 2.2). However, unlike in the

euthermic condition, a decrease in temperature did not significantly affect the affinity for

lactate of hibernator LDH as the temperature decreased (P>0.05, Table 2.2). The change in

temperature from 37°C to 5°C also lead to 0.21- and 0.11- fold changes in the Vmax values

in the lactate-oxidizing direction for euthermic and hibernator enzymes, respectively

(Table 2.2).

In the pyruvate-reducing direction, the affinity for pyruvate in euthermic and

hibernator LDH did not change with a decrease in temperature (Table 2.2). However, like

observed in the lactate-oxidizing direction, the change in temperature from 37°C to 5°C

lead to a decrease in Vmax values for the pyruvate-reducing direction. The change in Vmax

was 0.20- fold for euthermic LDH, and 0.08- fold for hibernator LDH (P

33

hibernator LDH as pH decreased; however, at 5°C there was no significant change in Vmax

values across both conditions with a change in pH (Table 2.3a,b).

In the pyruvate-reducing direction at 5°C, a decrease in pH did not affect pyruvate

affinity, reaction velocity, or inhibition by pyruvate in either euthermic or hibernating

conditions (Table 2.3a,b). However, at 37°C, a decrease in the pH lead to a significant

increase in pyruvate affinity, as well as a significant decrease in the IC50 of pyruvate for

both euthermic and hibernator forms of the enzyme (Table 2.3a,b).

34

Table 2.1. Purification and yield of U. richardsonii LDH from muscle of (a) euthermic and (b) hibernating ground squirrels.

35

Table 2.2. Kinetic parameters of purified muscle LDH from euthermic and hibernating U. richardsonii.

Enzyme parameter Euthermic Hibernating

Forward reaction (lactate→ pyruvate, pH 8.0) Km lactate, 37°C (mM) 5.21 ± 0.66 5.55 ± 0.79

Km lactate, 22°C (mM) 4.07 ± 0.31 3.19 ± 0.35

Km lactate, 5°C (mM) 2.67 ± 0.17b 4.87 ± 0.45a

Km NAD, 37°C (mM) 0.19 ± 0.02 0.14 ± 0.02

Km NAD, 22°C (mM) 0.12 ± 0.01b 0.06 ± 0.01b

Km NAD, 5°C (mM) 0.06 ± 0.01b 0.06 ± 0.01b

Activation Energy (kJ/mol) 34.52 ± 6.68 48.61 ± 13.19

Vmax, 37°C (mU/µg) 1.86 ± 0.30 4.91 ±0.46a

Vmax, 22°C (mU/µg) 0.72± 0.06b 1.11 ± 0.05a,b

Vmax, 5°C (mU/µg) 0.39 ± 0.05b 0.54 ± 0.09b

Reverse reaction (pyruvate→ lactate, pH 7.5) Km pyruvate, 37°C (mM) 0.13 ± 0.01 0.13 ± 0.01

Km pyruvate, 22°C (mM) 0.12 ± 0.01 0.12 ± 0.03

Km pyruvate, 5°C (mM) 0.09 ± 0.01 0.07 ± 0.01a

Activation Energy (kJ/mol) 35.10 ± 12.62 55.26 ± 15.69

Vmax, 37°C (mU/µg) 14.66 ± 2.33 26.97 ± 2.89a

Vmax, 22°C (mU/µg) 4.43 ± 0.32b 4.83 ± 0.57b

Vmax, 5°C (mU/µg) 2.93 ± 0.15b 2.19 ± 0.29a,b

IC50 pyruvate, 37°C (mM) 13.66 ± 1.46 15.16 ± 2.06

IC50 pyruvate, 22°C (mM) 7.52 ± 0.14b 4.42 ± 0.19a,b

IC50 pyruvate, 5°C (mM) 8.57 ± 1.16 9.71 ± 2.09

a indicates a significant difference from corresponding euthermic condition, Student’s t test, two-tailed, P

36

Table 2.3. Effects of pH on purified muscle LDH from (a) euthermic and (b) hibernating U. richardsonii.

Enzyme parameter (a) Euthermic (b) Hibernating

Forward reaction (lactate→ pyruvate) pH 8.0 pH 7.5 pH 8.0 pH 7.5

Km lactate, 37°C (mM) 5.21 ± 0.66 8.35 ± 1.17

5.55 ± 0.79 7.24 ± 0.91

Km lactate, 22°C (mM) 4.07 ± 0.31 2.87 ± 0.66 3.19 ± 0.35 5.27 ± 0.73

Km lactate, 5°C (mM) 2.67 ± 0.17 2.59 ± 0.35 4.87 ± 0.45 6.64 ± 0.39a

Km NAD, 37°C (mM) 0.19 ± 0.02 0.27 ± 0.05 0.14 ± 0.02 0.48 ± 0.08a

Km NAD, 22°C (mM) 0.12 ± 0.01 0.54 ± 0.06a 0.06 ± 0.01 0.76 ± 0.01

a

Km NAD, 5°C (mM) 0.06 ± 0.01 0.13 ± 0.01a 0.06 ± 0.01 0.15 ± 0.01

a

Activation Energy (kJ/mol) 34.52 ± 6.68 44.61 ± 4.67 48.61 ± 13.19 69.78 ± 18.79

Vmax, 37°C (mU/µg) 1.86 ± 0.30 2.84 ± 0.34

a 4.91 ±0.46 10.00 ± 0.96

a

Vmax, 22°C (mU/µg) 0.72± 0.06 0.97± 0.09a 1.11 ± 0.05 1.19 ± 0.11

Vmax, 5°C (mU/µg) 0.39 ± 0.05 0.38 ± 0.02 0.54 ± 0.09 0.42 ± 0.02

Reverse reaction (pyruvate→ lactate) pH 8.0 pH 7.5 pH 8.0 pH 7.5

Km pyruvate, 37°C (mM) 0.22 ± 0.03 0.13 ± 0.01a 0.28 ± 0.06 0.13 ± 0.01

a

Km pyruvate, 22°C (mM) 0.06 ± 0.01 0.12 ± 0.01a 0.29 ± 0.05 0.12 ± 0.03

a

Km pyruvate, 5°C (mM) 0.08 ± 0.01 0.09 ± 0.01 0.07 ± 0.01 0.07 ± 0.01

Activation Energy (kJ/mol) 4.87 ± 0.38 35.10 ± 12.62 47.76 ± 5.24 55.26 ± 15.69

Vmax, 37°C (mU/µg) 3.66 ± 0.67 14.66 ± 2.33

a 23.87 ± 1.55 26.97 ± 2.89

Vmax, 22°C (mU/µg) 3.27 ± 0.40 4.43 ± 0.32 7.58 ± 1.64 4.83 ± 0.57

Vmax, 5°C (mU/µg) 2.94 ± 0.40 2.93 ± 0.15 2.81 ± 0.12 2.19 ± 0.29

IC50 pyruvate, 37°C (mM) 24.32 ± 3.00 13.66 ± 1.46a 25.10 ± 3.28 15.16 ± 2.063

a

IC50 pyruvate, 22°C (mM) 12.23 ± 0.37 7.52 ± 0.14a 4.69 ± 0.58 4.42 ± 0.19

IC50 pyruvate, 5°C (mM) 9.29 ± 0.79 8.57 ± 1.16 8.42 ± 1.84 9.71 ± 2.09

a indicates a significant difference from pH 8.0 in each corresponding condition, Student’s t test, two tailed, P

37

Figure 2.1. Typical Cibacron Blue elution profiles for LDH activity from muscle of euthermic (control) and hibernating U. richardsonii using (a) 0-6 mM gradient of pyruvate + NADH and; (b) 0-2 M gradient of KCl as the elutant. Elution profiles are from separate runs of euthermic and hibernating enzyme but are superimposed here for viewing.

38

Figure 2.2. 10% SDS-PAGE gel with silver staining of samples taken at each step in the purification of LDH from muscle of euthermic U. richardsonii. Lanes (a) molecular weight ladder (FroggiaBio); (b) 1:100 diluted crude extract; (c) pooled LDH peak fractions after elution from the Cibacron blue agarose column with NADH+Pyruvate; (d) pooled LDH peak fractions from the Cibacron Blue agarose column after elution with KCl.

39

Figure 2.3. Effects of in vitro incubations to stimulate the activities of endogenous (a) protein kinases or (b) protein phosphatases on the Km for L-Lactate for LDH from euthermic U. richardsonii muscle. Crude extracts were incubated for 24 h before assay at 5⁰C. Data are means ± SEM, with at least n=3 separate determinations on enzyme isolated from different individuals. Conditions are defined in the Materials and Methods. Asterisks indicate significant differences from the ‘STOP’ condition, Student’s t-test, two-tailed, P < 0.05.

40

Figure 2.4. Quantification of post-translational modifications of purified LDH from muscle of euthermic and hibernating U. richardsonii. Chemiluminescence signal intensities were standardized to protein amount, and the value for hibernating LDH was expressed relative to the control value that was set to 1. Data are mean ± SEM, n=3-4 determinations on purified enzyme samples. Asterisks indicate significant differences from the corresponding control LDH, Student’s t-test, two-tailed, p

41

Figure 2.5. Michaelis-Menten curves for (a) forward (lactate-utilizing, 5˚C) and (b) reverse (pyruvate-utilizing, 5˚C) reactions catalyzed by purified euthermic and hibernating LDH; and (c) Arrhenius plots for the forward reaction LDH at three temperatures: 5˚C, 22˚C, 37˚C. Michaelis-Menten curves for euthermic LDH (black circles) and hibernating LDH (open circles). Plots are fitted with a three-parameter Hill coefficient curve using SigmaPlot 12. Data are mean ± SEM, n = 3-4 individual determinations on purified enzyme samples

42

Discussion

LDH is the terminal enzyme in anaerobic glycolysis; it facilitates the continuing

generation of ATP during oxygen deficient times by regenerating the NAD+ that is needed

to maintain glycolytic flux and producing an end product (lactate) that can be accumulated

and stored in high levels in tissues or transported for catabolism by other tissues

(Hochachka and Somero, 2014). Its role in the production of the important redox molecule

NAD+ makes LDH an interesting target for analysis. Like other small mammalian

hibernators, Richardson’s ground squirrels are capable of depressing their metabolism by

entering into cycles of torpor and arousal over the winter months. Despite the depression

of metabolism, these species must maintain vital physiological processes. With no food

intake during the winter hibernation months, these animals must reorganize their fuel

metabolism to maintain cell and tissue viability (Buck and Barnes, 2000). This

reorganization is essential not only for the maintenance of muscle energetics throughout

the hibernating season, but also critical for the arousal process since shivering

thermogenesis plays a major role in the restoration of euthermic Tb when animals arouse

from a torpor bout (Wang and Lee, 1996). The present chapter shows that skeletal muscle

LDH from euthermic versus hibernating animals displays different kinetic properties,

different responses to temperature, and appears to be regulated via reversible protein

phosphorylation.

i. LDH purification

Ground squirrel skeletal muscle LDH was purified to electrophoretic homogeneity

by using a combination of ion-exchange and affinity chromatography. The end result was

an enzyme preparation that was purified 29-fold to a specific activity of 11832 mU/mg

43

protein (Table 2.1; Fig 2.1). The fold purification and specific activity values for this LDH

are in accordance with the values obtained using the same three-step purification scheme

to purify LDH from the liver of Xenopus laevis (Katzenback et al., 2014). Thus, the results

presented here shed light on the reproducibility of the ion-exchange and affinity

chromatography purification scheme across different tissues and species and is the first of

its kind for a mammalian hibernator.

ii. Post-translational modification of LDH

Although LDH is known to have multiple isozymes (Markert and Møller, 1959),

skeletal muscle LDH is mainly a homotetramer of the muscle (M)-type subunit (LDH-5).

To date, no differential regulation of LDH subunit gene expression has been reported that

may contribute to the enzyme’s structural, functional, and/or regulatory properties in

euthermic versus hibernating states. Furthermore, the high energetic costs associated with

creating new proteins makes differential gene expression an unlikely event because during

hibernation ground squirrels need to be in an energy-conserving state. With that in mind,

structural and functional differences between euthermic and hibernating forms of LDH are

likely due to reversible post-translational modifications of the protein. In the present study,

the phosphorylation state of LDH was assessed through Western blot analysis using

phosphospecific antibodies. The analysis using anti-phospho-serine and anti-phospho-

threonine antibodies demonstrated that LDH from torpid animals showed significantly

greater serine and threonine phosphorylation as compared to euthermic conditions (Fig.

2.4). LDH has been found to be differentially phosphorylated in other species capable of

depressing their metabolism (Xiong and Storey, 2012; Dawson et al., 2013; Katzenback et

al., 2014). In Xiong and Storey (2012), the hypometabolic (anoxic) form of liver LDH

44

from anoxia-tolerant turtles showed greater phosphorylation as compared to the control

enzyme. The present data is consistent with the findings in turtles in that during hibernation

(a hypometabolic state in mammals) LDH has greater phosphorylation compared to the

euthermic enzyme. However, research conducted by Dawson et al. (2013) showed that

during stress, the anoxic form of turtle muscle LDH was, in fact, the low phosphate form.

The reversible phosphorylation differences seen in each of these studies may reflect

varying regulatory mechanisms across different species, or the need for tissue-specific

differences in LDH regulation. Despite differences in phosphorylation states, a similarity

between the current study and the previous ones described above is that the increase in

bound phosphate on LDH consistently decreased LDH activity in the lactate-oxidizing

direction.

To assess the effect of changes in phosphorylation state on the kinetic

characteristics of LDH, crude preparations of euthermic LDH were incubated under

conditions that would stimulate protein kinases or phosphatases and the Km of lactate was

assessed. Incubations that stimulated endogenous protein phosphatases caused a significant

decrease in the Km of lactate for euthermic LDH (Fig 2.3b). Furthermore, incubations that

investigated the role of specific protein kinases in altering LDH kinetics identified AMPK,

CaMK, and PKA as kinases that caused an approximately 2-fold increase in the Km of

lactate for control LDH (Fig. 2.3a). Each of these kinases has various roles in metabolism

and may potentially be important regulators of LDH in vivo (Storey and Storey, 2004a).

Most notably in vivo, LDH may be subject to AMPK control, AMPK being a kinase that

is highly sensitive to cellular nutrient/energy status as reflected in the [AMP] to [ATP] ratio

(reviewed in Hardie et al., 2006). Indeed, AMPK is often characterized as the energy sensor

45

or the cell. During fasting conditions, such as in hibernation, cellular [AMP] levels rise and

in turn, activate AMPK. This AMPK activation has profound effects on glycolytic activity

as it has been shown to down regulate the transcription of pyruvate kinase—a key

glycolytic enzyme (Kawaguchi et al., 2002). In this study, phosphorylation of LDH via

AMPK may serve as a complimentary way of ensuring that pyruvate levels stay minimal

during hibernation.

iii. LDH Kinetics

Kinetic analysis of LDH purified from euthermic and hibernating animals revealed

several differences between the two enzyme states. Properties of the enzymes were

analyzed at three temperatures, 37°C, 22°C, and 5°C, representing the approximate Tb

values that are reported in ground squirrels during euthermia, entrance in and out of torpor,

and during hibernation, respectively. A comparison between euthermic and hibernating

LDH revealed interesting findings. Most notably, at 5°C the Km of lactate was substantially

higher for hibernating LDH as compared to euthermic LDH (Table 2.2). Furthermore, at

this same temperature, Km of pyruvate was lower for hibernating LDH compared to the

euthermic form (Table 2.2). Previous research has shown that NAD+ concentrations in a

similar hibernator entering torpor are approximately 2 times higher than that found in their

active state (Serkova et al., 2007). The LDH kinetic data presented in the current study,

suggests that the hibernator form of LDH that is active at low Tb aids in the accumulation

of lactate and NAD+.

The activity of ground squirrel LDH showed a linear response to temperature

changes in vitro (Fig. 2.5c). Using pyruvate as a substrate, the activation energies of

euthermic (35.10 ± 12.62 kJ/mol) and hibernating LDH (55.26 ± 15.69 kJ/mol) were not

46

significantly different from one another (Table 2.2) and are similar to that of the Ea reported

for human LDH-5 (40.9 kJ/mol) (Tanishima et al., 1995). Studies of the effect of

temperature on Km for lactate revealed that a decrease in temperature from 37°C to 5°C

increased binding affinity for the substrate in euthermic LDH but did not affect the binding

affinity in the hibernating form (Table 2.2). This maintenance of Km of lactate aids in

suppressing the lactate-oxidizing reaction catalyzed by LDH at the low body temperatures

endured during torpor. Furthermore, and as expected, reaction velocities decreased

approximately 2-fold for every 10°C drop in temperature. The effect of pH on LDH activity

must also be considered as small changes in cytosolic pH may occur due to the effect of

low temperature on cell buffering systems during hibernation.

Studies looking at muscle pH in the Colombian ground squirrel revealed a slight

increase in cellular pH as the temperature decreased from 37°C (pH 7.24) to 5°C (pH 7.45)

(McArthur et al., 1990). With this in mind, it was appropriate to look at the effect of pH

on LDH activity in the current model. Although the increase in pH from 7.5 to 8.0 resulted

in a greater affinity for L-lactate at 5°C, the resulting Km at pH 8.0 was higher than the Km

values reported for euthermic LDH at either pH at 5°C (Figure 2.3). Therefore, at either

pH value at 5°C, hibernating LDH is more effective than its euthermic counterpart in

suppressing the lactate-oxidizing reaction.

iv. Conclusion

Purified skeletal muscle LDH from Richardson’s ground squirrels showed

distinctly different structural and kinetic properties between euthemic and hibernating

conditions, with hibernating LDH appearing to be less active in the lactate-oxidizing

direction at a low, hibernating temperature as compared to euthermic LDH. Given that

47

skeletal muscle cells are generally non-gluconeogenic and many glycolytic enzymes are

suppressed during hibernation, LDH modification may be necessary to prevent the

accumulation of pyruvate and help maintain NAD+ levels within muscle cells.

Chapter 3

Purification and Properties of Glycerol-3-

Phosphate Dehydrogenase from the Liver

of the Hibernating Ground Squirrel,

Urocitellus richardsonii

49

Introduction

To survive harsh winter climates, many small mammals are capable entering a

hypometabolic state, marked by an active suppression of their metabolic rate (MR) to limit

energy expenditures and a consequent lowering of their body temperature (Tb) (Lyman et

al., 2013). Mammals that use torpor are generally classified as either species using daily

torpor (i.e. daily heterotherms that drop their Tb by a few degrees Celsius during the

inactive part of their day) or multi-day hibernation. Daily heterotherms typically enter

torpor bouts lasting between approximately 3 and 12 hours, typically in a facultative

manner responding to current environmental temperature and food conditions. In contrast,

hibernators cycle through consecutive, prolonged periods (on average 1 week for ground

squirrels) of torpor and show a circannual rhythm where hibernation only occurs during

the winter season (Geiser and Ruf, 1995). Richardson’s ground squirrels (Urocitellus

richardsonii) ae seasonal hibernators that allow Tb to sink to near-ambient during winter

dormancy but defend a Tb about 5°C (using low levels thermogenesis by brown adipose) if

ambient temperature